Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice

Abstract

Background: Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.

Results: First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.

Conclusions: Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

Fig 1. Obif protein is O-glycosylated at serine residue 36.

(A) Potential O-glycosylation sites of human and mouse Obif proteins. The predicted amino acid sequences of human OBIF (NP_859075.2) and mouse Obif (NP_666274.1) were aligned by the ClustalW program (http://clustalw.ddbj.nig.ac.jp/). Asterisks indicate identical amino acids. Colons and periods indicate similar amino acids. Yellow boxes indicate potential O-glycosylation sites. Red bracket indicates extracellular domain. Blue underline indicates N-terminal signal peptides. Green box indicates single transmembrane domain. (B-C) Analysis of O-glycosylation sites in the mouse Obif protein. Constructs of pCAGGS expression vector (CAG), FLAG-tagged GFP (GFP), or FLAG-tagged mObif with or without mutation(s) (wild-type (WT), S36A, S43A, T54A, T60A, T67A, or S36A/S43A/T54A/T60A/T67A (ALL)) were transfected into HEK293T cells. The HEK293T cells were cultured for 24 h. The cell lysates were analyzed by Western blotting using an anti-FLAG M2 antibody (B). FLAG-tagged constructs expressing GFP (GFP), human CD55 (hCD55), or wild-type mouse Obif (mObif-WT) were transfected into HEK293T cells cultured in standard medium. The HEK293T cells were cultured for 24 h, and subsequently cultured for 3 days in medium with or without benzyl-GalNAc. The cell lysates were analyzed by Western blot analysis using the anti-FLAG M2 antibody (C). Arrowheads indicate the 60 kDa band of O-glycosylated mObif. Arrows indicate the 37 kDa band is a nascent form of mObif-WT. Benzyl-GalNAc, benzyl 2-acetamido-2-deoxy- α-D-galactopyranoside, an O-glycosylation inhibitor.

Fig 2. Loss of Obif impaired bone growth.

(A) CR lengths from wild-type (white box) and Obif ^−/− mice (black box) at 8 wks. (B) Longitudinal bone lengths of radius, humerus, tibia, and femur in wild-type (white box) and Obif ^−/− mice (black box) at 8 wks. CR length, crown-rump length. Error bars show the SEM (n = 5). *P < 0.05.

Fig 3. Histological analysis of distal femoral epiphysis of Obif ^−/− mice.

(A-B) Villanueva bone staining of distal femur sections from wild-type and Obif ^−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and Obif ^−/− mice (black box) (A). In wild-type and Obif ^−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm (A) and 20 μm (B). Error bars show the SEM (n = 3).

Fig 4. Obif ^−/− mice showed abnormal bone formation and bone mineralization.

(A) Fluorescence microscopic images of calcein (indicated by arrowhead) and tetracycline (indicated by arrow) staining of distal femur sections from wild-type and Obif ^−/− mice. Scale bar represents 100 μm. (B-R) Bone histomorphometric analyses of the distal femur sections from wild-type (white box) and Obif ^−/− mice (black box) at 8 wks. Parameters for bone formation (B-E), bone resorption (F-I), bone volume (J-M), and mineralization (N-R) were analyzed. OV/BV, osteoid volume per bone volume; OV/OS, osteoid volume per osteoid surface; Ob.S/BS, osteoblast surface per bone surface; N.Ob/BS, osteoblast number per bone surface; ES/BS, eroded surface per bone surface; Oc.S/BS, osteoclast surface per bone surface; N.Oc/BS, osteoclast number per bone surface; BRs.R, bone resorption rate; BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; MAR, mineral apposition rate; BFR/BS, bone formation rate per bone surface; Aj.Ar, adjusted MAR; Omt, osteoid maturation time; Mlt, mineralization lag time. Error bars show the SEM (n = 5). *P < 0.05.

Fig 5. Obif is required for normal spermatogenesis.

(A) Gross appearance of testes in male wild-type and Obif ^−/− mice at 12 wks. (B) Comparison of testicular weight in wild-type (white box), Obif ^+/− (grey box), and Obif ^−/− (black box) mice at 5 wks and 12 wks (n = 6). (C) Comparison of epididymis weight between wild-type and Obif ^−/− mice at 12 wks (n = 6). (D) Comparison of sperm number from cauda epididymis between wild-type, Obif ^+/−, and Obif ^−/− at 16 wks and 24 wks (n = 4). (E) The level of serum testosterone in wild-type and Obif ^−/− mice at 12 wks (n = 6). (F) H&E staining of testis sections from wild-type and Obif ^−/− mice at 12 wks. Scale bars represent 5 mm (A), and 50 μm (F). Error bars show the SEM. *P < 0.05.

Fig 6. Late spermatogenesis is perturbed in the Obif ^−/− testis.

(A-E) In situ hybridization analysis for testicular sections from wild-type and Obif ^−/− mice at 12wks, using probes of PLZF (A), Acrosin (B), Calmegin (C), 3β-HSD (D), and Sox9 (E). (F) Immunofluorescence staining of testicular sections with an anti-SPACA1 antibody, which detects cells at stages from spermatids to spermatozoa. Arrows, round spermatids; arrowheads, elongated spermatids; asterisks, spermatozoa. Scale bars represent 100 μm (A-E) and 20 μm (F).